CN117285617B - Recombinant metallothionein Pro.MT and preparation method and application thereof - Google Patents
Recombinant metallothionein Pro.MT and preparation method and application thereof Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/825—Metallothioneins
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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- A—HUMAN NECESSITIES
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
- A61P39/06—Free radical scavengers or antioxidants
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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Abstract
The invention obtains a recombinant metallothionein Pro.MT by a site-directed mutagenesis mode, which is characterized in that the recombinant metallothionein MT has 3 mutation sites E5C, M9C, K C on the basis of human metallothionein-4. Through site-directed mutagenesis, cys residue number is increased at a specific position of the beta-domain fragment, so that a new tetrametallosulfur cluster can be formed by the beta-domain in the E5C, M9C, K C mutant, the acid stability of the protein is improved, the difference of the beta-domain and the alpha-domain in the acid stability and the thiol reactivity is eliminated, and the oxidation resistance of the metallosulfur protein is further enhanced. The invention also constructs a multi-copy tandem expression vector of Pro.MT, which is formed by connecting more than two copies in series, a section of flexible joint region is included between the copies, the recombinant metallothionein expression vector can efficiently express the polymetallic metallothionein, each metallothionein monomer has a complete and independent characteristic structural domain, and the expression product has higher acid stability and high oxidation resistance.
Description
Technical Field
The invention relates to a recombinant metallothionein Pro.MT, a dimer thereof, a coding gene, an expression vector, an expression host bacterium, a preparation method and application thereof, and belongs to the technical field of protein expression.
Background
The Metallothionein (MTs) is a low molecular weight cysteine-rich metal binding protein, and has unique functions in the aspects of antioxidation, free radical removal, in-vivo necessary metal element balance maintenance, heavy metal toxicity removal and the like due to the fact that the cysteine-rich residues (more than 30%) are combined with metal ions in a reduced state and the like.
Human metallothionein is divided into four subgroups: MT1, MT2, MT3, MT4.MT1 and MT2 are distributed throughout the body, MT3 is distributed in the brain, and MT4 is mainly distributed in the skin.
Free radicals are the first murder of human aging, are closely related to a plurality of middle-aged and elderly diseases of human beings, so that the immunity of the human beings is gradually reduced, metallothionein is the best known free radical scavenger at present, and MT is the only effective heavy metal detoxification and scavenging protein at present.
The existing metallothionein is mainly derived from animal viscera extract, has the problems of small extraction amount, high preparation cost, partial heavy metal pollution and the like, and influences the wide application of the metallothionein.
Disclosure of Invention
The invention aims at providing a recombinant metallothionein Pro.MT, which belongs to mutants of human metallothionein MT4 subgroup.
The invention adopts the technical scheme that:
recombinant metallothionein Pro.MT, the sequence of which is shown in SEQ ID No. 1; or has more than 95 percent of homology with SEQ ID No.1, and the in vitro free radical scavenging capacity is basically equivalent to the protein shown in SEQ ID No. 1.
The coding gene of the recombinant metallothionein Pro.MT has a sequence shown in SEQ ID No. 3.
The expression vector of the recombinant metallothionein Pro.MT.
Expression host bacteria of the recombinant metallothionein Pro.MT.
The preparation method of the recombinant metallothionein Pro.MT comprises the following steps:
(1) Constructing the expression vector and transforming the expression vector into expression host bacteria;
(2) Culturing an expression host bacterium, and inducing the expression of recombinant metallothionein Pro.MT;
(3) Purifying the expression product to obtain the recombinant metallothionein Pro.MT.
The invention also discloses a recombinant metallothionein Pro.MT dimer which is a dimer composed of two recombinant metallothionein Pro.MT monomers, and the two monomers are connected through a joint.
Preferably, the sequence is shown in SEQ ID No. 2.
The coding gene of the recombinant metallothionein Pro.MT dimer has a sequence shown in SEQ ID No. 4.
Expression vector of recombinant metallothionein Pro.MT dimer.
Expression host bacteria of the recombinant metallothionein Pro.MT dimer.
The use of the recombinant metallothionein Pro.MT described above or the recombinant metallothionein Pro.MT dimer described above for scavenging free radicals in vitro.
The invention has the beneficial effects that:
the invention obtains a recombinant metallothionein Pro.MT by a site-directed mutagenesis mode, which is characterized in that the recombinant metallothionein MT has 3 mutation sites E5C, M9C, K C on the basis of human metallothionein-4. By site-directed mutagenesis, the addition of Cys residues at specific positions in the β -domain fragment allows β -domain to form a new tetra-metallothionein in the E5C, M9C, K C mutant, which is encapsulated by the peptide fragment in a manner that is more compact than that of the wild-type β -domain. The novel tetrametallosulfur cluster generation eliminates the difference of beta-and alpha-domains in acid stability and sulfhydryl reactivity, and further enhances the oxidation resistance of metallosulfur proteins.
The invention also constructs a recombinant metallothionein multi-copy tandem expression vector which is formed by connecting more than two copies in series, a section of flexible joint region is arranged between the copies, the recombinant metallothionein expression vector can efficiently express the poly-metallothionein, each metallothionein monomer has a complete and independent characteristic structural domain, and the expression product has higher acid stability and high oxidation resistance.
Drawings
FIG. 1 is a plasmid map of recombinant metallothionein Pro.MT dimer.
Detailed Description
The invention is further illustrated by the following examples, which are not intended to be limiting. Specific materials and sources thereof used in embodiments of the present invention are provided below. However, it should be understood that these are merely exemplary and are not intended to limit the present invention, as materials that are the same as or similar to the type, model, quality, nature, or function of the reagents and instruments described below may be used in the practice of the present invention. The experimental methods used in the following examples are conventional methods unless otherwise specified. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Example 1
The construction method of the recombinant metallothionein Pro.MT expression vector comprises the following steps:
(1) The amino acid sequence (shown as SEQ ID NO. 1) of the recombinant metallothionein Pro.MT is reversely translated into a DNA coding sequence, and the coding DNA sequence (shown as SEQ ID NO. 3) is obtained by optimizing the sequence of a ribosome binding region through codon preference, but the sequence is not limited to the coding DNA sequence, and the limitation is that the homology of the amino acid sequence of the translated protein is equal to or more than 85 percent. The pMV-Pro.MT plasmid containing the Pro MT DNA fragment shown in SEQ ID NO.3 was synthesized by Beijing Liuhua macrogene technology Co.
(2) And (3) designing a primer through Snap Gene software by using the synthesized Pro.MT coding sequence, amplifying a Pro.MT gene fragment by using a PCR technology, recovering a PCR product, connecting the Pro.MT gene to a vector pET30a by using a homology arm primer and adopting a Gibson connection technology to construct the expression vector pET30a-Pro.MT. The ligation product was transformed into E.coli DH 5. Alpha. Competent cells, positive transformants were selected by kanamycin resistance, agarose gel electrophoresis verification was performed on the selected transformants, and positive transformants were selected for sequencing verification and plasmid extraction.
TABLE 1PCR amplification System
| System components | Volume of the composition |
| Primer F | 2.5μL |
| Primer R | 2.5μL |
| Q5 High-Fidelity 2x Master Mix | 2μL |
| pMV-Pro.MT plasmid containing Pro MT DNA fragment | 25μL |
| ddH2O | To 50μL |
(3) After the PCR system is prepared, the mixture is evenly mixed and centrifuged, and the PCR amplification conditions are as follows: the first stage is pre-denaturation at 98 ℃ for 30s; the second stage of denaturation at 98 ℃ for 10s, annealing at 50-72 ℃ for 30s, extension at 72 ℃ for 30s/kb,32 cycles; the third stage extends at 72 deg.C for 2min. The above vectors and gene fragments were recovered using a universal DNA purification kit (Tiangen Biochemical Co., ltd.) and carried out according to the procedure of the product instruction.
TABLE 2Gibson ligation System
Mixing above components on ice, and heating at 37deg.C for 60min to obtain a connection product, and storing on ice or at-20deg.C for subsequent competent transformation.
(4) Transformation of E.coli DH 5. Alpha. Competent cells: slowly thawing competent bacteria on ice (the competent bacteria can be directly used for fresh preparation), adding 3-5ul of the connection product, flicking a centrifuge tube with a light finger to mix the bacteria and the plasmid connection product, standing in an ice bath for 30min, performing heat shock in a water bath at 42 ℃ for 2min after the completion, adding 500ul of LB, performing shaking culture at a constant temperature of 37 ℃ and 220rpm for 1 hour, centrifuging 2000-3000g for 1min after culture to precipitate thalli, leaving about 50-100ul of supernatant to resuspend and precipitate, then coating all on an LB plate containing ampicillin resistance, and performing inversion culture at 37 ℃ for overnight.
By the same method, a wild type human metallothionein-4 monomer hMT4 expression vector pET30a-hMT4 was constructed as a control, wherein the Gene ID of hMT 4: 84560, source https:// www.ncbi.nlm.nih.gov/gene/84560.
Example 2
The pET30a-Pro.MT dimer tandem expression vector was constructed in substantially the same manner as in the examples. The amino acid sequence of the Pro/MT dimer is shown as SEQ ID NO.2, the optimized coding DNA sequence is shown as SEQ ID NO.4, and the constructed expression vector is named as pET30 a-Pro/MT-Linker 1-Pro/MT mutant.
By adopting the same method, a wild type human metallothionein-4 (hMT 4) dimer tandem expression vector pET30a-hMT4-Linker1-hMT4 is constructed as a control, wherein the hMT4 dimer is formed by connecting two hMT4 monomers through a GGGGSGGGGSGGGGS joint.
Example 3 protein expression test
The pET30a-Pro.MT, pET30a-hMT4, pET30a-Pro.MT-Linker1-Pro.MT, pET30a-hMT4-Linker1-hMT4 vector plasmids obtained in examples 1 and 2 were transformed into competent cells of E.coli BL21 (DE 3), and positive transformants were selected for kanamycin resistance.
Picking single colony, culturing in LB culture medium, inducing with IPTG for 20 hr, centrifugally collecting thallus, washing with 1XPBS for 2 times, re-suspending in 1XPBS, ultrasonically crushing thallus, centrifugally collecting supernatant.
Mixing 20uL of supernatant with loading buffer solution, heating at 95 ℃ for 10 minutes, centrifuging, instantly separating to the bottom of a tube, and placing on ice.
10uL of 15% polyacrylamide is used for electrophoresis, coomassie brilliant blue is used for dyeing and decoloring, and the expression conditions of protein bands, metallothionein 4 contrast and mutants are observed.
Human metallothionein-4 monomer hMT4, 62aa total, molecular weight 6.5kDa, accounting for 38% of total protein of the thallus, soluble expression;
Pro.MT, 62aa total, molecular weight 6.5kDa, accounting for 36% of total protein of the thallus, soluble expression;
hMT4 dimer, 155aa total, molecular weight 15.4kDa, accounting for 40% of total protein of thallus, soluble expression;
Pro.MT dimer, 155aa total, molecular weight 15.4kDa, 42% of total protein of the cell, soluble expression. The specific data are shown in Table 3.
TABLE 3 protein expression test results
Example 4 in vitro radical clearance assay
1, 1-diphenyl-2-picrylhydrazine free radical (DPPH) is the main material for researching in vitro free radical elimination, is a stable free radical in ethanol, and has a purple solution and shows the maximum absorption peak at 517 nm. When DPPH encounters a proton donor substance, such as an antioxidant, the color changes from purple to yellow, and the absorbance decreases, whereby the radical scavenging ability of the chemical can be determined.
The recombinant metallothionein 4 control and the ability of the mutant monomer to scavenge free radicals in vitro with the dimer solution were determined by incubating 0.1mL of hMT4 monomer and dimer obtained by expression of example 3 (25, 50, 100, 200, 400. Mu.g/mL), pro.MT monomer and dimer solution (the solution refers to supernatant after induction of expression in example 3, diluted to the corresponding concentration by Elisa assay) and 0.1mL of 80. Mu.g/mL DPPH solution with the same volume of ultrapure water as a control, with an enzyme-labeled instrument at 517nm wavelength after incubation for 30 minutes in the absence of light, and calculating IC50 (the concentration of solution required for DPPH clearance of 50%).
TABLE 4 results of testing the DPPH-scavenging ability of different proteins
Claims (10)
1. A recombinant metallothionein Pro.MT is characterized in that the sequence is shown in SEQ ID No. 1.
2. The recombinant metallothionein Pro.MT of claim 1 encoding gene.
3. The coding gene according to claim 2, wherein the sequence is shown in SEQ ID No. 3.
4. The recombinant metallothionein Pro.MT expression vector of claim 1.
5. The method for preparing recombinant metallothionein Pro.MT according to claim 1, which is characterized by comprising the following steps:
(1) Constructing the expression vector of claim 4, and transforming the expression vector into expression host bacteria;
(2) Culturing an expression host bacterium, and inducing the expression of recombinant metallothionein Pro.MT;
(3) Purifying the expression product to obtain the recombinant metallothionein Pro.MT according to claim 1.
6. A recombinant metallothionein Pro.MT dimer, which is a dimer composed of two recombinant metallothionein Pro.MT monomers according to claim 1, wherein the two monomers are connected by a linker.
7. Recombinant metallothionein Pro.MT dimer according to claim 6, characterised in that it has the sequence shown in SEQ ID No. 2.
8. The recombinant metallothionein Pro.MT dimer encoding gene of claim 7, wherein the sequence is shown in SEQ ID No. 4.
9. The recombinant metallothionein Pro.MT dimer expression vector of claim 7.
10. Use of the recombinant metallothionein pro.mt of claim 1 or the recombinant metallothionein pro.mt dimer of claim 7 for scavenging free radicals in vitro.
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